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Mutations in DVL1 Cause an Osteosclerotic Form of Robinow Syndrome

2015; Elsevier BV; Volume: 96; Issue: 4 Linguagem: Inglês

10.1016/j.ajhg.2015.02.010

1537-6605

Kieran J. Bunn, Phil Daniel, Heleen S. Rösken, Adam C. O’Neill, Sophia Cameron‐Christie, Tim Morgan, Han G. Brunner, Angeline Lai, Henricus P. M. Kunst, David Markie, Stephen P. Robertson,

Cancer-related gene regulation

Robinow syndrome (RS) is a phenotypically and genetically heterogeneous condition that can be caused by mutations in genes encoding components of the non-canonical Wnt signaling pathway. In contrast, germline mutations that act to increase canonical Wnt signaling lead to distinctive osteosclerotic phenotypes. Here, we identified de novo frameshift mutations in DVL1, a mediator of both canonical and non-canonical Wnt signaling, as the cause of RS-OS, an RS subtype involving osteosclerosis, in three unrelated individuals. The mutations all delete the DVL1 C terminus and replace it, in each instance, with a novel, highly basic sequence. We showed the presence of mutant transcript in fibroblasts from one individual with RS-OS and demonstrated unimpaired protein stability with transfected GFP-tagged constructs bearing a frameshift mutation. In vitro TOPFlash assays, in apparent contradiction to the osteosclerotic phenotype, revealed that the mutant allele was less active than the wild-type allele in the canonical Wnt signaling pathway. However, when the mutant and wild-type alleles were co-expressed, canonical Wnt activity was 2-fold higher than that in the wild-type construct alone. This work establishes that DVL1 mutations cause a specific RS subtype, RS-OS, and that the osteosclerosis associated with this subtype might be the result of an interaction between the wild-type and mutant alleles and thus lead to elevated canonical Wnt signaling. Robinow syndrome (RS) is a phenotypically and genetically heterogeneous condition that can be caused by mutations in genes encoding components of the non-canonical Wnt signaling pathway. In contrast, germline mutations that act to increase canonical Wnt signaling lead to distinctive osteosclerotic phenotypes. Here, we identified de novo frameshift mutations in DVL1, a mediator of both canonical and non-canonical Wnt signaling, as the cause of RS-OS, an RS subtype involving osteosclerosis, in three unrelated individuals. The mutations all delete the DVL1 C terminus and replace it, in each instance, with a novel, highly basic sequence. We showed the presence of mutant transcript in fibroblasts from one individual with RS-OS and demonstrated unimpaired protein stability with transfected GFP-tagged constructs bearing a frameshift mutation. In vitro TOPFlash assays, in apparent contradiction to the osteosclerotic phenotype, revealed that the mutant allele was less active than the wild-type allele in the canonical Wnt signaling pathway. However, when the mutant and wild-type alleles were co-expressed, canonical Wnt activity was 2-fold higher than that in the wild-type construct alone. This work establishes that DVL1 mutations cause a specific RS subtype, RS-OS, and that the osteosclerosis associated with this subtype might be the result of an interaction between the wild-type and mutant alleles and thus lead to elevated canonical Wnt signaling. Robinow syndrome (RS [dominant RS, MIM 180700; recessive RS, MIM 268310]) is a genetically and phenotypically heterogeneous skeletal dysplasia characterized by the distinctive facial appearance of midface hypoplasia, hypertelorism, a short nose, and a broad mouth, known collectively as "fetal facies." Additional but variable features of RS include mesomelic dwarfism, macrocephaly, gingival hypertrophy, dental malocclusion, genital hypoplasia, brachydactyly, bifid thumbs, and segmentation defects (reviewed in Mazzeu et al.1Mazzeu J.F. Pardono E. Vianna-Morgante A.M. Richieri-Costa A. Ae Kim C. Brunoni D. Martelli L. de Andrade C.E. Colin G. Otto P.A. Clinical characterization of autosomal dominant and recessive variants of Robinow syndrome.Am. J. Med. Genet. A. 2007; 143: 320-325Crossref PubMed Scopus (72) Google Scholar). Some forms of RS are caused by mutations in genes encoding components of Wnt signaling pathways.2Afzal A.R. Rajab A. Fenske C.D. Oldridge M. Elanko N. Ternes-Pereira E. Tüysüz B. Murday V.A. Patton M.A. Wilkie A.O. Jeffery S. Recessive Robinow syndrome, allelic to dominant brachydactyly type B, is caused by mutation of ROR2.Nat. Genet. 2000; 25: 419-422Crossref PubMed Scopus (240) Google Scholar, 3van Bokhoven H. Celli J. Kayserili H. van Beusekom E. Balci S. Brussel W. Skovby F. Kerr B. Percin E.F. Akarsu N. Brunner H.G. Mutation of the gene encoding the ROR2 tyrosine kinase causes autosomal recessive Robinow syndrome.Nat. Genet. 2000; 25: 423-426Crossref PubMed Scopus (216) Google Scholar, 4Person A.D. Beiraghi S. Sieben C.M. Hermanson S. Neumann A.N. Robu M.E. Schleiffarth J.R. Billington Jr., C.J. van Bokhoven H. Hoogeboom J.M. et al.WNT5A mutations in patients with autosomal dominant Robinow syndrome.Dev. Dyn. 2010; 239: 327-337PubMed Google Scholar Wnt signaling is a complex pleiotropic network for which more than 19 Wnt ligands and more than 15 Wnt receptors and co-receptors have been described.5Niehrs C. The complex world of WNT receptor signalling.Nat. Rev. Mol. Cell Biol. 2012; 13: 767-779Crossref PubMed Scopus (972) Google Scholar Signaling via these complexes is broadly divided into canonical and non-canonical pathways. Activation of the canonical pathway by Wnt ligands prevents the degradation of β-catenin, which accumulates in the cytoplasm and moves to the nucleus to alter gene expression.5Niehrs C. The complex world of WNT receptor signalling.Nat. Rev. Mol. Cell Biol. 2012; 13: 767-779Crossref PubMed Scopus (972) Google Scholar Germline mutations in the canonical pathway in humans cause a number of defects, the most notable of which is an impact upon bone mineral density (BMD). A high-bone-mass phenotype (MIM 144750) is caused by gain-of-function mutations in LRP5 (MIM 603506),6Little R.D. Carulli J.P. Del Mastro R.G. Dupuis J. Osborne M. Folz C. Manning S.P. Swain P.M. Zhao S.C. Eustace B. et al.A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait.Am. J. Hum. Genet. 2002; 70: 11-19Abstract Full Text Full Text PDF PubMed Scopus (1084) Google Scholar, 7Boyden L.M. Mao J. Belsky J. Mitzner L. Farhi A. Mitnick M.A. Wu D. Insogna K. Lifton R.P. High bone density due to a mutation in LDL-receptor-related protein 5.N. Engl. J. Med. 2002; 346: 1513-1521Crossref PubMed Scopus (1332) Google Scholar whereas a reduction in expression or activity of the extracellular soluble Wnt antagonist SOST (MIM 605740) leads to van Buchem disease (MIM 239100)8Balemans W. Patel N. Ebeling M. Van Hul E. Wuyts W. Lacza C. Dioszegi M. Dikkers F.G. Hildering P. Willems P.J. et al.Identification of a 52 kb deletion downstream of the SOST gene in patients with van Buchem disease.J. Med. Genet. 2002; 39: 91-97Crossref PubMed Scopus (574) Google Scholar and sclerosteosis type 1 (MIM 269500).9Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. et al.Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein.Am. J. Hum. Genet. 2001; 68: 577-589Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar That an increase in canonical Wnt signaling will cause an increase in bone mass, and the converse, is well established through a number of other human10Gong Y. Slee R.B. Fukai N. Rawadi G. Roman-Roman S. Reginato A.M. Wang H. Cundy T. Glorieux F.H. Lev D. et al.Osteoporosis-Pseudoglioma Syndrome Collaborative GroupLDL receptor-related protein 5 (LRP5) affects bone accrual and eye development.Cell. 2001; 107: 513-523Abstract Full Text Full Text PDF PubMed Scopus (1851) Google Scholar, 11Leupin O. Piters E. Halleux C. Hu S. Kramer I. Morvan F. Bouwmeester T. Schirle M. Bueno-Lozano M. Fuentes F.J. et al.Bone overgrowth-associated mutations in the LRP4 gene impair sclerostin facilitator function.J. Biol. Chem. 2011; 286: 19489-19500Crossref PubMed Scopus (224) Google Scholar, 12Boudin E. Steenackers E. de Freitas F. Nielsen T.L. Andersen M. Brixen K. Van Hul W. Piters E. A common LRP4 haplotype is associated with bone mineral density and hip geometry in men-data from the Odense Androgen Study (OAS).Bone. 2013; 53: 414-420Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar and animal13Kato M. Patel M.S. Levasseur R. Lobov I. Chang B.H. Glass 2nd, D.A. Hartmann C. Li L. Hwang T.H. Brayton C.F. et al.Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor.J. Cell Biol. 2002; 157: 303-314Crossref PubMed Scopus (941) Google Scholar, 14Kokubu C. Heinzmann U. Kokubu T. Sakai N. Kubota T. Kawai M. Wahl M.B. Galceran J. Grosschedl R. Ozono K. Imai K. Skeletal defects in ringelschwanz mutant mice reveal that Lrp6 is required for proper somitogenesis and osteogenesis.Development. 2004; 131: 5469-5480Crossref PubMed Scopus (147) Google Scholar studies (reviewed in Wang et al.15Wang Y. Li Y.P. Paulson C. Shao J.Z. Zhang X. Wu M. Chen W. Wnt and the Wnt signaling pathway in bone development and disease.Front Biosci (Landmark Ed). 2014; 19: 379-407Crossref PubMed Scopus (178) Google Scholar). Non-canonical Wnt signaling is more mechanistically heterogeneous in that it involves a number of interrelated signaling cascades. Germline mutations in genes encoding components of one particular non-canonical Wnt signaling pathway, termed the planar cell polarity (PCP)-Wnt pathway, cause some forms of RS.2Afzal A.R. Rajab A. Fenske C.D. Oldridge M. Elanko N. Ternes-Pereira E. Tüysüz B. Murday V.A. Patton M.A. Wilkie A.O. Jeffery S. Recessive Robinow syndrome, allelic to dominant brachydactyly type B, is caused by mutation of ROR2.Nat. Genet. 2000; 25: 419-422Crossref PubMed Scopus (240) Google Scholar, 3van Bokhoven H. Celli J. Kayserili H. van Beusekom E. Balci S. Brussel W. Skovby F. Kerr B. Percin E.F. Akarsu N. Brunner H.G. Mutation of the gene encoding the ROR2 tyrosine kinase causes autosomal recessive Robinow syndrome.Nat. Genet. 2000; 25: 423-426Crossref PubMed Scopus (216) Google Scholar, 4Person A.D. Beiraghi S. Sieben C.M. Hermanson S. Neumann A.N. Robu M.E. Schleiffarth J.R. Billington Jr., C.J. van Bokhoven H. Hoogeboom J.M. et al.WNT5A mutations in patients with autosomal dominant Robinow syndrome.Dev. Dyn. 2010; 239: 327-337PubMed Google Scholar RS has both dominantly and recessively inherited forms, which differ both by their phenotype and by their mode of inheritance.1Mazzeu J.F. Pardono E. Vianna-Morgante A.M. Richieri-Costa A. Ae Kim C. Brunoni D. Martelli L. de Andrade C.E. Colin G. Otto P.A. Clinical characterization of autosomal dominant and recessive variants of Robinow syndrome.Am. J. Med. Genet. A. 2007; 143: 320-325Crossref PubMed Scopus (72) Google Scholar Recessive RS, characterized by severe mesomelia and segmentation defects such as hemivertebrae or rib fusions, is caused by biallelic loss-of-function mutations in the gene encoding the transmembrane Wnt co-receptor ROR2 (receptor tyrosine kinase-like orphan receptor 2).2Afzal A.R. Rajab A. Fenske C.D. Oldridge M. Elanko N. Ternes-Pereira E. Tüysüz B. Murday V.A. Patton M.A. Wilkie A.O. Jeffery S. Recessive Robinow syndrome, allelic to dominant brachydactyly type B, is caused by mutation of ROR2.Nat. Genet. 2000; 25: 419-422Crossref PubMed Scopus (240) Google Scholar, 3van Bokhoven H. Celli J. Kayserili H. van Beusekom E. Balci S. Brussel W. Skovby F. Kerr B. Percin E.F. Akarsu N. Brunner H.G. Mutation of the gene encoding the ROR2 tyrosine kinase causes autosomal recessive Robinow syndrome.Nat. Genet. 2000; 25: 423-426Crossref PubMed Scopus (216) Google Scholar In a small minority of individuals with dominant RS, characterized by a milder phenotype without rib fusions, the condition is caused by loss-of-function mutations in the gene encoding the classical PCP-Wnt ligand Wnt-5a.4Person A.D. Beiraghi S. Sieben C.M. Hermanson S. Neumann A.N. Robu M.E. Schleiffarth J.R. Billington Jr., C.J. van Bokhoven H. Hoogeboom J.M. et al.WNT5A mutations in patients with autosomal dominant Robinow syndrome.Dev. Dyn. 2010; 239: 327-337PubMed Google Scholar No other mutations have previously been associated with human RS. It is well known that there is substantial cross-talk between the canonical and non-canonical pathways (reviewed in Niehrs5Niehrs C. The complex world of WNT receptor signalling.Nat. Rev. Mol. Cell Biol. 2012; 13: 767-779Crossref PubMed Scopus (972) Google Scholar). Despite this, there is no indication that the Mendelian conditions discussed above have impacts across more than one Wnt pathway. However, a number of Wnt mediators are shared components of both canonical and non-canonical signaling—the dishevelled family of proteins (DVL) is the primary example.16Gao C. Chen Y.G. Dishevelled: The hub of Wnt signaling.Cell. Signal. 2010; 22: 717-727Crossref PubMed Scopus (542) Google Scholar Three unrelated individuals with the clinical diagnosis of sporadic RS with atypical dramatic osteosclerosis (RS-OS) were ascertained and described previously (Figures 1A–1G).17Bunn K.J. Lai A. Al-Ani A. Farella M. Craw S. Robertson S.P. An osteosclerotic form of Robinow syndrome.Am. J. Med. Genet. A. 2014; 164A: 2638-2642Crossref PubMed Scopus (9) Google Scholar, 18Eijkenboom D.F. Verbist B.M. Cremers C.W. Kunst H.P. Bilateral conductive hearing impairment with hyperostosis of the temporal bone: a new finding in Robinow syndrome.Arch. Otolaryngol. Head Neck Surg. 2012; 138: 309-312Crossref PubMed Scopus (5) Google Scholar These individuals exhibited a clinical presentation of dominant RS with characteristic facial dysmorphism (marked hypertelorism, short nose, broad mouth, and midface hypoplasia), camptodactyly and brachydactyly (in subjects 1 and 2), cleft palate (in subjects 2 and 3), and dental anomalies. Mesomelia, another common finding in RS, ranged from mild to absent in these individuals, but this observation does not preclude a clinical diagnosis of RS.19Bain M.D. Winter R.M. Burn J. Robinow syndrome without mesomelic 'brachymelia': a report of five cases.J. Med. Genet. 1986; 23: 350-354Crossref PubMed Scopus (41) Google Scholar Radiographs revealed osteosclerosis of the cranium, which was particularly prominent at the skull base (Figure 1E). In subjects 1 and 2, generalized axial and appendicular osteosclerosis was also documented and was particularly marked in the cortices of the long bones, which were also undertubulated (Figures 1F and 1G). No information on the remainder of the skeleton was available for individual 3. In subjects 1 and 2, the distal phalanges of the hands and feet were hypoplastic, and the terminal phalanx of the thumb was bifid, a pattern typical of RS.20Al Kaissi A. Bieganski T. Baranska D. Chehida F.B. Gharbi H. Ghachem M.B. Hendaoui L. Safi H. Kozlowski K. Robinow syndrome: report of two cases and review of the literature.Australas. Radiol. 2007; 51: 83-86Crossref PubMed Scopus (11) Google Scholar Dual-energy X-ray absorptiometry scanning of the skeleton revealed markedly elevated BMD (lumbar spine Z scores were +7.4 for subject 1 and +7.6 for subject 2). The parents of all three individuals were clinically unremarkable. Table 1 summarizes these clinical features.Table 1Clinical and Radiographic Features of the Three Subjects with RS-OSSubject 1Subject 2Subject 3Hypertelorism+++Mesomelia−−−Cleft palate−++Camptodactyly and brachydactyly++−Gingival hyperplasia++−Oligodontia+++Bilateral mixed hearing loss+++Osteosclerosis of skull+++Osteosclerosis of long bones++NABifid thumb and great toe++NAAbbreviations are as follows: +, present; −, absent; NA, information not available. Open table in a new tab Abbreviations are as follows: +, present; −, absent; NA, information not available. Elevated BMD occurring in the context of sporadic RS in three unrelated individuals of different ethnic backgrounds and without relevant family history, consanguinity, or sibling recurrence led us to hypothesize that this phenotype was underpinned by rare de novo mutations at a previously unidentified locus. All subjects participated after informed consent was obtained in keeping with the principles of the Declaration of Helsinki. Ethical approval for this work was obtained from the Southern Health and Disability Ethics Committee of New Zealand (reference no. 13/STH/56). Accordingly, we performed whole-exome sequencing and employed a parent-proband-trio design to identify de novo variants in subject 1. DNA was extracted from blood leukocytes and captured with an Agilent SureSelect All Exon V4 Kit and sequenced on an Illumina HiSeq2000 with paired-end 100-bp reads. Data were processed for alignment, and variants were called according to the current best-practice guidelines from the Broad Institute.21Van der Auwera G.A. Carneiro M.O. Hartl C. Poplin R. Del Angel G. Levy-Moonshine A. Jordan T. Shakir K. Roazen D. Thibault J. et al.From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline.Curr. Protoc. Bioinformatics. 2013; 11 (33): 1-11Google Scholar Reads were aligned to the human genome (GRCh37) with the Burrows-Wheeler Aligner (BWA)-MEM,22Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26628) Google Scholar base quality scores were recalibrated with the Genome Analysis Toolkit (GATK), and variants were called with the GATK HaplotypeCaller. Each exome had an average read depth of >30×. Variants were annotated with SnpEff,23Cingolani P. Platts A. Wang L. Coon M. Nguyen T. Wang L. Land S.J. Lu X. Ruden D.M. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3.Fly (Austin). 2012; 6: 80-92Crossref PubMed Scopus (5592) Google Scholar and SnpSift was used to identify de novo mutations in subject 1.24Cingolani P. Patel V.M. Coon M. Nguyen T. Land S.J. Ruden D.M. Lu X. Using Drosophila melanogaster as a Model for Genotoxic Chemical Mutational Studies with a New Program, SnpSift.Front. Genet. 2012; 3: 35Crossref PubMed Scopus (515) Google Scholar Novelty was determined by the exclusion of variants identified in 400 in-house exomes and of variants found in the NHLBI Exome Sequencing Project Exome Variant Server (ESP6500). Variants were filtered for quality and for a read depth of >5× across the trio. Annotation with SnpEff was used to filter for effects likely to disrupt protein sequences. All apparently de novo single-base substitutions and small insertions or deletions were validated by Sanger sequencing of the parents and proband. Two coding variants, a frameshift (c.1519del [p.Trp507Glyfs∗142]; RefSeq accession number NM_004421.2) in DVL1 (MIM 601365) and a single-base substitution (c.2753C>T [p.Pro918Leu]; RefSeq NM_014494.2) in TNRC6A (MIM 610739), satisfied these criteria as the only functional de novo mutations found in this individual. TNRC6A encodes a component of a cytoplasmic ribonucleoprotein complex that regulates mRNA silencing, stability, and translation. PolyPhen-2,25Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. Kondrashov A.S. Sunyaev S.R. A method and server for predicting damaging missense mutations.Nat. Methods. 2010; 7: 248-249Crossref PubMed Scopus (9284) Google Scholar which uses physical properties and evolutionary conservation to predict deleterious effects of coding variants, predicted that the p.Pro918Leu substitution is likely to be benign. No novel sequence variants in TNRC6A were observed in subject 2. DVL1 is a central mediator of Wnt signal transduction. The c.1519del frameshift falls within the penultimate exon of the 15-exon DVL1. This mutation is predicted to remove almost the entirety of the C-terminal tail of DVL1 and replace this sequence with a 142-amino-acid-long, novel, highly basic sequence (the predicted pI of the C-terminal section rises from 9.5 to 12.526Wilkins M.R. Gasteiger E. Bairoch A. Sanchez J.C. Williams K.L. Appel R.D. Hochstrasser D.F. Protein identification and analysis tools in the ExPASy server.Methods Mol. Biol. 1999; 112: 531-552PubMed Google Scholar). Direct sequencing of the relevant exons and intron-exon boundaries of DVL1 in individuals 2 and 3 identified two similar de novo frameshift mutations in DVL1: c.1562del (p.Pro521Hisfs∗128) and c.1576_1583delinsG (p.Pro526Alafs∗121), respectively. Each DVL1 mutation leads to a frameshift in the same reading frame. All three DVL1 mutations are predicted to lead to a protein product 23 residues shorter than wild-type DVL1 (Figures 1H–1J). The same novel C-terminal sequence, which ranges from 121 to 142 residues in length and differs only in the extent of its N terminus, is predicted to be appended to the DVL1 proteins produced from these mutant alleles (Figures 1I and 1J). DVL proteins are widely conserved Wnt signaling mediators throughout evolution.27Dillman A.R. Minor P.J. Sternberg P.W. Origin and evolution of dishevelled.G3 (Bethesda). 2013; 3: 251-262Crossref PubMed Scopus (14) Google Scholar Mammals have three DVL1 paralogs (DVL1–DVL3), all of which share a high level of sequence homology. They encode intracellular scaffolding proteins acting directly downstream from the transmembrane Wnt receptors (frizzled family). At DVL, the Wnt pathways functionally diverge into the canonical and non-canonical cascades; which way they diverge is dependent upon which downstream mediators DVL interacts with (reviewed in Gao and Chen16Gao C. Chen Y.G. Dishevelled: The hub of Wnt signaling.Cell. Signal. 2010; 22: 717-727Crossref PubMed Scopus (542) Google Scholar). The three DVL paralogs have separate but overlapping roles, which are elegantly shown in a series of mouse knockouts.28Wynshaw-Boris A. Dishevelled: in vivo roles of a multifunctional gene family during development.Curr. Top. Dev. Biol. 2012; 101: 213-235Crossref PubMed Scopus (43) Google Scholar Of the three Dvl knockouts, loss of Dvl1 has the least phenotypic impact; null mice display no dysmorphic phenotype.29Lijam N. Paylor R. McDonald M.P. Crawley J.N. Deng C.X. Herrup K. Stevens K.E. Maccaferri G. McBain C.J. Sussman D.J. Wynshaw-Boris A. Social interaction and sensorimotor gating abnormalities in mice lacking Dvl1.Cell. 1997; 90: 895-905Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar In contrast, Dvl2-null mice have cardiac, segmentation, and neural-tube defects.30Hamblet N.S. Lijam N. Ruiz-Lozano P. Wang J. Yang Y. Luo Z. Mei L. Chien K.R. Sussman D.J. Wynshaw-Boris A. Dishevelled 2 is essential for cardiac outflow tract development, somite segmentation and neural tube closure.Development. 2002; 129: 5827-5838Crossref PubMed Scopus (377) Google Scholar Dvl3-null mice show pathologies similar to those of Dvl2-null mice, and additional cochlear anomalies, and rarely survive until adulthood.31Etheridge S.L. Ray S. Li S. Hamblet N.S. Lijam N. Tsang M. Greer J. Kardos N. Wang J. Sussman D.J. et al.Murine dishevelled 3 functions in redundant pathways with dishevelled 1 and 2 in normal cardiac outflow tract, cochlea, and neural tube development.PLoS Genet. 2008; 4: e1000259Crossref PubMed Scopus (241) Google Scholar DVL proteins have three well-defined functional domains: the N-terminal DIX (dishevelled and axin), the PDZ (postsynaptic density 95, disc large, and zonula occludens 1), and the C-terminal DEP (dishevelled, Egl-10, and pleckstrin) domains (Figure 1J). To date, no mutations at any of the DVL loci have been associated with clinically apparent defects in human Wnt signaling. The three clustered mutations in the individuals described in this study fall outside the three established domains of DVL and thus introduce C-terminal frameshifts after the DEP domain. However, a number of conserved C-terminal regions, including sites that are subject to phosphorylation,32González-Sancho J.M. Greer Y.E. Abrahams C.L. Takigawa Y. Baljinnyam B. Lee K.H. Lee K.S. Rubin J.S. Brown A.M. Functional consequences of Wnt-induced dishevelled 2 phosphorylation in canonical and noncanonical Wnt signaling.J. Biol. Chem. 2013; 288: 9428-9437Crossref PubMed Scopus (51) Google Scholar binding sites for ubiquitin ligases,33Miyazaki K. Fujita T. Ozaki T. Kato C. Kurose Y. Sakamoto M. Kato S. Goto T. Itoyama Y. Aoki M. Nakagawara A. NEDL1, a novel ubiquitin-protein isopeptide ligase for dishevelled-1, targets mutant superoxide dismutase-1.J. Biol. 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Han W.H. Lee J.H. Cho J.Y. Park W.S. Maurice M.M. Han J.K. Lee M.J. Finley D. Jho E.H. Deubiquitination of Dishevelled by Usp14 is required for Wnt signaling.Oncogenesis. 2013; 2: e64Crossref PubMed Scopus (81) Google Scholar the binding site for IQGAP1 (which controls DVL nuclear localization38Goto T. Sato A. Shimizu M. Adachi S. Satoh K. Iemura S. Natsume T. Shibuya H. IQGAP1 functions as a modulator of dishevelled nuclear localization in Wnt signaling.PLoS ONE. 2013; 8: e60865Crossref PubMed Scopus (25) Google Scholar), and a frizzled interacting domain.39Tauriello D.V. Jordens I. Kirchner K. Slootstra J.W. Kruitwagen T. Bouwman B.A. Noutsou M. Rüdiger S.G. Schwamborn K. Schambony A. Maurice M.M. Wnt/β-catenin signaling requires interaction of the Dishevelled DEP domain and C terminus with a discontinuous motif in Frizzled.Proc. Natl. Acad. Sci. USA. 2012; 109: E812-E820Crossref PubMed Scopus (145) Google ScholarConsidering the lack of malformations in Dvl1−/− mice, the lack of phenotypic effects similar to RS-OS in individuals with deletions encompassing DVL1,40Firth H.V. Richards S.M. Bevan A.P. Clayton S. Corpas M. Rajan D. Van Vooren S. Moreau Y. Pettett R.M. Carter N.P. DECIPHER: Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources.Am. J. Hum. Genet. 2009; 84: 524-533Abstract Full Text Full Text PDF PubMed Scopus (1167) Google Scholar and our observation that all three frameshift mutations replace functional domains with the same, highly basic novel C terminus, these DVL1 alleles seem to confer the RS-OS phenotype by a specific mechanism distinct from simple haploinsufficiency. Thus, the focus of this work was to understand the mechanistic basis of the osteosclerotic aspect of the RS-OS phenotype, given that osteosclerosis is the main phenotypic feature that differentiates these individuals from other subjects with RS. The three DVL1 mutations all occur within a confined region of DVL1 and could be acting through either a loss or gain of function. All three fall in the penultimate exon, where the novel reading frame does not encounter a premature stop codon before entering the last exon (Figures 1I and 1J). This observation presents the possibility that these mutation-bearing transcripts could escape nonsense-mediated decay (NMD). To investigate, we prepared cDNA from dermal fibroblasts obtained from subject 1 and performed RT-PCR to amplify a product that spanned parts of exons 14 and 15, including the site of the mutation. After this product was sequenced, both mutant and wild-type alleles were observed on the Sanger chromatogram, suggesting that the mutant allele was not subject to NMD (data not shown). To confirm this, we digested these same RT-PCR products with BstN1, which specifically cuts at a site that is destroyed by the mutation. A substantial proportion of the product derived from the subject-1 cDNA was refractory to digestion, whereas a control was digested to completion (Figure 2A). To ascertain whether the frameshift-containing protein produced from the c.1519del allele found in individual 1 demonstrated significant instability, we performed an immunoblot on skin fibroblast lysate, but this failed to detect DVL1 in either subject or control cultures. To circumvent this inability to detect endogenous DVL1, we cloned an N-terminal GFP-tagged full-length DVL1 construct (GFP-wtDVL1) and introduced the c.1519del mutation by site-directed mutagenesis (GFP-mtDVL1). This clone was transfected into C2C12 cells, and immunodetection was performed with anti-GFP antibodies with band intensities normalized to endogenous GAPDH. These results indicated no apparent difference in abundance between the altered and wild-type DVL1 (Figure 2B). This was confirmed by infrared immunofluorescence, normalized to α-tubulin (n = 3; Figure S1). These data indicate that mutation-bearing alleles that lead to RS-OS are transcribed and translated into stable proteins and consequently hold the potential to exert novel biological functions or interfere with endogenous DVL1-mediated activities. Previous work has found that loss of the DVL1 C terminus reduces the protein's ability to signal in the canonical Wnt pathway and is mediated by either a loss of interaction with the frizzled receptor39Tauriello D.V. Jordens I. Kirchner K. Slootstra J.W. Kruitwagen T. Bouwman B.A. Noutsou M. Rüdiger S.G. Schwamborn K. Schambony A. Maurice M.M. Wnt/β-catenin signaling requires interaction of